Background & objective: RNA interference (RNAi) technique is now widely used in studies of gene function, signal transduction pathway, and gene therapy because it can effectively and specifically inhibit gene expression. This study was designed to synthesize small interfering RNA (siRNA) by in vitro transcription, and construct retrovirus vectors to express small hairpin RNA (shRNA), detect RNAi in nasopharyngeal carcinoma cell lines, and to develop a RNAi technique platform.
Methods: siRNAs targeting green fluorescent protein (GFP) and luciferase (Luc) were synthesized by in vitro transcription, while shRNAs targeting GFP and Luc were constructed from pSUPER.retro. Cervical cancer cell line HeLa, nasopharyngeal carcinoma cell lines CNE1, CNE2, and 5-8F were co-transfected with siRNAs or shRNAs and reporter gene pEGFP-N1 or pGL3. The expression of GFP was detected by fluorescent microscopy and Western blot. The activity of luciferase was measured by Luciferase Enzyme Assay System.
Results: siRNA duplexes with 3' UU overhangs and shRNA specifically silenced GFP expression, while antisense RNA and siRNA without 3' UU overhangs did not trigger RNA interference of GFP. Quantitative luciferase activity analysis showed that siRNA inhibited Luc expression in HeLa, CNE1, CNE2, and 5-8F cell lines with inhibition rates of 91.43%, 78.01%, 90.30%, and 62.85%, respectively. Similarly, the inhibition rate was 78.22% when shRNA targeting Luc was co-transfected into HeLa cell line.
Conclusions: Both siRNAs and shRNAs can induce RNAi. 3' UU overhangs of siRNA may play a role in RNAi. RNAi can be triggered in both nasopharyngeal carcinoma cell lines and HeLa cell line.